Familial Non-Autoimmune Hyperthyroidism Caused by an Extracellular Domain Variant (p.Leu267Phe) of the TSH Receptor

Non-autoimmune hyperthyroidism (NAH) is a rare genetic disorder caused by germline-activating variants in the TSH receptor (TSHR) gene. While most NAH-related TSHR variants are located in the seven-transmembrane domain (7TMD), three variants (p.Leu267Phe, p.Ser281Asn, and p.Asn406Ser) have been identified in the extracellular domain (ECD), with p.Leu267Phe previously not showing constitutively active in vitro.

We searched for TSHR variants in a Japanese family with NAH using PCR-based direct sequencing. We created three ECD variants (p.Leu267Phe, p.Ser281Asn, and p.Asn406Ser) and a series of variants in which Leu267 was mutated to 18 amino acids other than Leu and Phe. Based on the cryo-electron microscopic structures, we evaluated the structure-function relationship of TSHR. We compared their cAMP-producing capacities to WT-TSHR in the luciferase activity assay using HEK293 cells. Western blot and fluorescence immunostaining were performed using HA-tagged TSHR vectors to compare Leu267Phe-TSHR with WT-TSHR.

A heterozygous TSHR variant (p.Leu267Phe) was identified. Comparison of the cryo-electron microscopy structures of the activated and inactivated TSHRs revealed a significant change in the ECD structure around Leu267. The ligand-independent cAMP-producing capacities compared to WT-TSHR were 238 ± 20% (mean ± SEM) for Leu267Phe. Of all 19 possible variants created through systematic mutagenesis, only Leu267Phe-TSHR and Leu267Tyr-TSHR exhibited significantly higher ligand-independent cAMP-producing capacities. Immunoblotting and fluorescence immunostaining showed that the Leu267Phe variant did not affect protein expression levels and intracellular localization of TSHR.

Leu267Phe-TSHR causes NAH. Substituting Leu267 to aromatic amino acids may shift the equilibrium of the TSHR state toward activation.